Cell viability assays are used by many researchers to evaluate cell health. This assay category is useful for experimental optimization, understanding cell metabolism, studying the effects of a certain treatment, etc. In this article, we will specifically focus on the ATP bioluminescence assay, which uses the relationship between ATP, luciferin and luciferase to help determine the number of live cells.
The ATP bioluminescence assay assumes that living cells in a sample have a constant amount of ATP. And it uses that assumption along with the chemical relationship between ATP, luciferin and luciferase in order to measure cell viability. In other words, it allows researchers to measure the amount of living or dead cells within a sample based on the presence or absence of ATP.
In the case of a living cell, which has ATP, the luciferin-luciferase reaction would produce a bioluminescent flash. In the case of a dead cell, which does not have ATP, no bioluminescence reaction would occur.The amount of signal intensity, under healthy conditions, is constant for each living cell. Therefore, researchers can use the bioluminescence intensity to understand the overall number of living cells within a sample (Brovko, 2010).
Figure 1. Illustration showing the assumption that
live cells within a sample will produce a constant amount of ATP. Because the reaction requires ATP, the signal intensity will be proportional to the amount of ATP in the sample.
The ATP bioluminescence assay works by exposing a sample of cells to the enzyme luciferase and the substrate luciferin. Because the luciferase reaction relies on ATP, only living cells in the sample would produce a bioluminescent signal.
Alternatively, in the case of dead cells, cell metabolism is disrupted. However, ATPases (enzymes that degrade ATP) still function. This leads to a quick drop in ATP in dead cells within the sample.
But to really understand how the ATP bioluminescence assay works, we need to understand the luciferase reaction.
There are two steps in the luciferase reaction. The first step is the adenylation of the substate luciferin which uses ATP. The second step is the oxidative decarboxylation of the luciferyl adenylate to form oxyluciferin. The oxyluciferin formed is at an excited state, and light is produced when it moves back to the ground state (Belanger & University of Rochester Introductory Biochemistry, 2018).
Figure 2. Shows the basic firefly luciferase reaction involving ATP and D-luciferin. Once the excited oxyluciferin reaches ground state, light is produced.
What this shows us is that ATP is a fundamental compound within the luciferase reaction. Knowing this, and the fact that ATP exists within living cells, researchers are able to utilize this reaction and get visual data about the number of living and dead cells within a sample.
The ATP bioluminescence assay is used for many studies such as (Riss et al., 2016), (Brovko, 2010):
- Sanitation evaluation
- Food health and food processing
- Guide to biocide dosing
- Managing fermentation
- Cell culture optimization
- Experimental condition optimization
- Treatment efficacy evaluation
- Understanding cytotoxic effects
- Measuring receptor binding
- Understanding signal transduction events
- Studying pathogenicity
- Oncology research
- Drug development
Many popular cell viability assays are considered invasive. They ultimately kill the cells. One of the biggest advantages of the ATP bioluminescence assay is that it is noninvasive.
The assay is not toxic and therefore does not destroy living cells. Furthermore, it allows real-time monitoring (Brovko, 2010).
Additional advantages of the ATP Bioluminescence Assay (AIB Staff, 2013):
- Simple to perform
- Extremely sensitive
- Very cost-effective
- Faster than conventional methods
- Results within minutes
The ATP bioluminescence assay relies on the luciferase reaction. Therefore, the substrates used will be firefly luciferin.
Keep in mind, there are other bioluminescent cell viability assays that use different substrates and compounds.
Cell viability assays using bacterial luciferase result in an autofluorescence. This method does not require an outside substrate, which can make it more advantageous than the firefly luciferase based method.
Renilla and Gaussia based cell viability assays use the substrate coelenterazine. One of the advantages to this method is that Gaussia is secreted from the cell, making it easier for in vivo detection and high-throughput screening. However, a disadvantage is that it’s outside of spectral range (480 nm), meaning the max wavelength emitted is outside of the boundaries of what would be in living tissue samples (Brovko, 2010).
While luciferin does easily permeate the eukaryotic cell membrane, challenges arise when dealing with the cell wall of a bacterial cell. This is because in solution luciferin is negatively charged.
One way around this is to lower the pH to a slightly acidic level. Doing so allows protonation of the carboxylic group of luciferin. Be careful not to lower the pH below 5.0, which will inactivate luciferase.
Another method of luciferin introduction into bacterial cells is by using polymyxin B. Polymyxin B creates pores in E. coli cells that allow luciferin to penetrate the cell. Again, there is a note of caution because you do not want to use a lethal concentration.
Finally, caged luciferins are another way to introduce luciferin into a bacterial cell. These modified substrates are neutral and are therefore more permeable. Upon entrance into the cell they can be converted into the active form either by an enzymatic process or by photoactivation (Brovko, 2010).
AIB Staff. (2013, October 11). A Q&A on ATP Bioluminescence assay. Retrieved March 22, 2021, from https://www.qualityassurancemag.com/article/aib1013-atp-bioluminescence-assay/
Belanger, S., & University of Rochester Introductory Biochemistry (Producers). (2018, May 15). Luciferase [Video file]. Retrieved March 22, 2021, from https://www.youtube.com/watch?v=odN92KbH8Bo
Brovko, L. (2010). Bioluminescence and fluorescence for in vivo imaging: In vivo optical imaging (Vol. TT91). Bellingham, WA, Washington: SPIE Press.
Riss, T., Moravec, R., Niles, A., Duellman, S., Benink, H., Worzella, T., & Minor, L. (2016). Cell Viability Assays. The Assay Guidance Manual.